The most important player in the calcium induced calcium release process is the "cardiac" ryanodine receptor, RyR2. This is a large protein (500 kD) which forms a tetrameric channel 30x30x15 nm in size. The trans-membrane channel is believed to be formed by the C-terminal. The bulk of the molecule forms the large "foot process" that spans the diadic cleft between the SR and sarcolemmal membranes; its function is unknown. We plan to study the in-vivo function of this channel by a strategy of producing a null background by (tissue restricted and inducible) knockout of the wild-type gene, followed by rescue with genetically altered components. Global manipulation of the cardiac excitation-contraction coupling machinery (e.g., developing an RyR2 null myocyte in which mutated RyR2 can be expresses) produces an embryonic-lethal phenotype. The goal is to induce a spatially and temporally dependent RyR2 knock-ou in mouse heart that can be rescued by mutated forms of the human RyR2 cDNA. To avoid embryonic lethality, we are taking the following approach: Conditional and inducible gene targeting, limited to specific cardiac lineages (e.g. ventricular myocytes) and inducible at a desired developmental stage (particularly in the adult). The tools to accomplish this are the Cre recombinase - LoxP recombination system and the tetracycline trans-activator system. A mouse is constructed which carries a Cre recombinase transgene under control of a tetracycline-sensitive promoter as well as a knockout construct containing LoxP sites in such a manner that induction of Cre expression by withdrawal of tetracycline causes excision of a critical exon of the target gene. This system is placed under control of a lineage-specific promoter (such as the ventricular myosin light chain MLC2V), so that a tissue-specific knockout can be made to occur at a specified time. Currently a number of founder lines containing a construct of tetop-Cre Recombinase have been identified. These lines are currently being studied for appropriate expression. A construct of MLC2V-tTA has been prepared and injected into the appropriate oocytes. In the case of the RyR2 knockout, a 15 kb mouse 129/SvJ genomic DNA fragment has been cloned, sequenced and confirmed to contain 4 exons of RyR2. The mutant mouse RyR2 targeting vector has been prepared in which RyR2 exons contain two flanking loxP sites and a pGK-neo resistant positive selection cassette as well as a pGK-tk negative selection cassette. The RyR2 gene targeting construct has been introduced into embryonic stem cells to establish inducible RyR2 functional channel knock-out mice to study the in vivo funciton of ryanodine receptor in ventricular myocardium. These cells are currently injected for identification of appropriate chimeras.